Stealth oxime ether lipid vesicles promote delivery of functional DsiRNA in human lung cancer A549 tumor bearing mouse xenografts.

We previously reported that hydroxylated oxime ether lipids (OELs) efficiently deliver functional Dicer substrate siRNAs (DsiRNAs) in cells. Here, we explored in vivo utility of these OELs, using OEL4 as a prototype and report that surface modification of the OEL4 formulations was essential for their in vivo applications. These surface-modified OEL4 formulations were developed by inclusion of various PEGylated lipids. The vesicle stability and gene knock-down were dependent on the PEG chain length. OEL4 containing DSPE-PEG350 and DSPE-PEG1000 (surprisingly not DSPE2000) promoted gene silencing in cells. In vivo studies demonstrated that OEL4 vesicles formulated using 3 mol% DSPE-PEG350 accumulate in human lung cancer (A549-luc2) xenografts in mice and exhibit a significant increase in tumor to liver ratios. These vesicles also showed a statistically significant reduction of luciferase signal in tumors compared to untreated mice. Taken together, the scalable OEL4:DSPE-PEG350 formulation serves as a novel candidate for delivery of RNAi therapeutics.

Identification of a reciprocal negative feedback loop between tau-modifying proteins MARK2 kinase and CBP acetyltransferase.

The posttranslational regulation of the neuronal proteome is critical for brain homeostasis but becomes dysregulated in the aged or diseased brain, in which abnormal posttranslational modifications (PTMs) are frequently observed. While the full extent of modified substrates that comprise the "PTM-ome" are slowly emerging, how the upstream enzymes catalyzing these processes are regulated themselves is not well understood, particularly in the context of neurodegeneration. Here, we describe the reciprocal regulation of a kinase, the microtubule affinity-regulating kinase 2 (MARK2), and an acetyltransferase, CREB-binding protein (CBP), two enzymes known to extensively modify tau proteins in the progression of Alzheimer's disease. We found that MARK2 negatively regulates CBP and, conversely, CBP directly acetylates and inhibits MARK2 kinase activity. These findings highlight a reciprocal negative feedback loop between a kinase and an acetyltransferase, which has implications for how PTM interplay is coordinated on substrates including tau. Our study suggests that PTM profiles occur through the posttranslational control of the master PTM remodeling enzymes themselves.

Photoactivation of sulfonated polyplexes enables localized gene silencing by DsiRNA in breast cancer cells.

Translation potential of RNA interference nanotherapeutics remains challenging due to in vivo off-target effects and poor endosomal escape. Here, we developed novel polyplexes for controlled intracellular delivery of dicer substrate siRNA, using a light activation approach. Sulfonated polyethylenimines covalently linked to pyropheophorbide-α for photoactivation and bearing modified amines (sulfo-pyro-PEI) for regulated endosomal escape were investigated. Gene knock-down by the polymer-complexed DsiRNA duplexes (siRNA-NPs) was monitored in breast cancer cells. Surprisingly, sulfo-pyro-PEI/siRNA-NPs failed to downregulate the PLK1 or eGFP proteins. However, photoactivation of these cell associated-polyplexes with a 661-nm laser clearly restored knock-down of both proteins. In contrast, protein down-regulation by non-sulfonated pyro-PEI/siRNA-NPs occurred without any laser treatments, indicating cytoplasmic disposition of DsiRNA followed a common intracellular release mechanism. Therefore, sulfonated pyro-PEI holds potential as a unique trap and release light-controlled delivery platform for on-demand gene silencing bearing minimal off target effects.